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RNA Immunoprecipitation (RIP)
RNA Immunoprecipitation (RIP) is a powerful method to study the physical relationship between proteins and RNA molecules in vivo. RIP is similar to ChIP, but instead precipitates RNA binding proteins (RBP), also known as ribonucleoproteins. The approach is based on the use of a specific antibody that is raised against the target protein to pull down the RBP and target-RNA complexes. Any RNA associated with the target protein can be isolated as well, making RIP exceptionally useful as a bioinformatic tool to help map protein-binding sites relating to mRNA processing and its regulation within the cell.
In a typical experiment, either cross-linked or native RNA-protein complexes can be used. The complexes are isolated by immunoprecipitation (IP) using a specific antibody towards the protein of interest. After reversing the cross-links, the interacting RNA can be analyzed by reverse transcription PCR (rt-PCR), genomic analysis using microarrays (RIP-Chip), or next-generation sequencing (NGS) methods (RIP-Seq).
There exist two main classes of RIP techniques: native or crosslinked. In both methods, the RNA is then extracted and subsequently analyzed by PCR techniques, microarray hybridization, and/or NGS.
Native RIP provides researchers the ability to reveal the identity of RNAs directly bound by the target protein, as well as determine the precise abundance in the IP sample. Experimentally, first harvested cells are directly lysed then IP is performed using an antibody raised against the target protein.
Crosslinked RIP, the second technique, allows the user to precisely map the direct and/or indirect binding site of the RBP of interest to the RNA molecule. Experimentally, first live cells are treated and fixed by formaldehyde or another cross-linking agent, like UV light. After a nuclear extraction step, the chromatin is sheared and the DNA is degraded through a DNase treatment. IP is performed and a reverse cross-linking step is incorporated to release the precipitated RNA.
In a typical experiment, either cross-linked or native RNA-protein complexes can be used. The complexes are isolated by immunoprecipitation (IP) using a specific antibody towards the protein of interest. After reversing the cross-links, the interacting RNA can be analyzed by reverse transcription PCR (rt-PCR), genomic analysis using microarrays (RIP-Chip), or next-generation sequencing (NGS) methods (RIP-Seq).
There exist two main classes of RIP techniques: native or crosslinked. In both methods, the RNA is then extracted and subsequently analyzed by PCR techniques, microarray hybridization, and/or NGS.
Native RIP provides researchers the ability to reveal the identity of RNAs directly bound by the target protein, as well as determine the precise abundance in the IP sample. Experimentally, first harvested cells are directly lysed then IP is performed using an antibody raised against the target protein.
Crosslinked RIP, the second technique, allows the user to precisely map the direct and/or indirect binding site of the RBP of interest to the RNA molecule. Experimentally, first live cells are treated and fixed by formaldehyde or another cross-linking agent, like UV light. After a nuclear extraction step, the chromatin is sheared and the DNA is degraded through a DNase treatment. IP is performed and a reverse cross-linking step is incorporated to release the precipitated RNA.
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Product Ordering Information
Table 1. RNA quantification and PCR reagents
| Product Name ▲ ▼ | Ex (nm) ▲ ▼ | Em (nm) ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
| StrandBrite™ Green Fluorimetric RNA Quantitation Kit *Optimized for Microplate Readers* | 490 nm | 545 nm | 1000 Tests | 17655 |
| StrandBrite™ Green Fluorimetric RNA Quantitation Kit | 490 nm | 540 nm | 100 Tests | 17656 |
| StrandBrite™ Green Fluorimetric RNA Quantitation Kit *High Selectivity* | 490 nm | 540 nm | 100 Tests | 17657 |
| StrandBrite™ Green RNA Quantifying Reagent | 490 nm | 525 nm | 1 mL | 17610 |
| StrandBrite™ Green RNA Quantifying Reagent | 490 nm | 525 nm | 10 mL | 17611 |
| Portelite™ Fluorimetric RNA Quantitation Kit | 490 nm | 525 nm | 100 Tests | 17658 |
| Portelite™ Fluorimetric RNA Quantitation Kit | 490 nm | 525 nm | 500 Tests | 17659 |
| Cyber Green™ [Equivalent to SYBR® Green] *20X Aqueous PCR Solution* | 498 nm | 522 nm | 5 x 1 mL Tests | 17591 |
| Cyber Green™ [Equivalent to SYBR® Green] *20X Aqueous PCR Solution* | 498 nm | 522 nm | 1 mL | 17592 |
| Cyber Green™ Nucleic Acid Gel Stain [Equivalent to SYBR® Green] | 498 nm | 522 nm | 100 µL | 17604 |